The biology of Anaplasma phagocytophilum (Ap) and its host are intertwined. Specific co-adaptations which have co-evolved over millennia govern the interactions between disease agents and humans. This proposal will address the symbiosis of parasite and host at the molecular level using human cells and a human pathogen. To understand how Ap exploits its host cells requires an understanding of what the host provides for the microbes. Our central hypothesis is that Ap redirects the cellular metabolic machinery for its own use. We hypothesize that Ap engages in molecular parasitism to complement its streamlined genome, that is deficient in many metabolic pathways. Therefore, we propose to determine the role of human host cells in supporting Ap parasitism through systematic knockdown of 47,400 human genes using a pseudoviral library of short interfering RNA (siRNA), followed by transfeetion of cells with specific siRNA constructs. Silencing expression of cellular genes critical for Ap infection will render cells unable to support Ap. We will conduct a genomic screen using an siRNA library to identify host factors required for adhesion, invasion, and growth of Ap in human cells. Host cells with gene knockdown(s) that interfere with Ap infection can be isolated, and the siRNA species responsible identified using human genomic DNA arrays. To evaluate the resultant list of genes, we will map them to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database to link the silenced genes to metabolic pathways. Selected genes identified on microarrays will be targeted using the pMaleficent Sleeping Beauty transposon based RNAi delivery system to produce heritable integration and expression of specific siRNA in human host cell lines. The ability of Ap to enter and multiply in host cells will be assessed by light and fluorescent microscopy, time-lapse microscopy, FACS analysis and by using a fluorescent plate reader. We expect to generate human cell lines with defined and stable gene knockdown phenotypes. Remapping affected genes on the KEGG database will reveal and confirm specific dependencies of Ap on human metabolites that may lead to new treatments and vaccines against these and other intracellular pathogens. Although the human genome has been sequenced, a significant proportion of ORFs are of unknown function. We anticipate that the interaction of the human host cells with these highly specialized intracellular pathogens will shed light,on the nature of some unidentified human genes. [unreadable] [unreadable] [unreadable]